P803: CRIP1 INDUCES PIS RESISTANCE BY FORMING A COMPLEX WITH USP7/PA200 AND ENHANCING PROTEASOME ACTIVITY AND AUTOPHAGY IN MULTIPLE MYELOMA
Authors: Zhen Yu, Peixia Tang, Zhenshu Xu, Lugui Qiu, Mu Hao
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple myeloma (MM) is a plasma malignancy characterized by an accumulation of misfolded immunoglobulins in the cell. Although proteasome inhibitor (PI) can treat MM by inhibiting ubiquitin-proteasome system (UPS), autophagy as a compensatory protein clearance mechanism, it still leads to drug resistance. The discovery of new targets that can simultaneously inhibit UPS and autophagy will provide great potential for the treatment of MM. Aims: In this study, we described CRIP1 expression patterns, and explored the inhibition effect of CRIP1 knockdown on ubiquitin-mediated proteasome degradation and autophagy. The mechanism involved in CRIP1-mediated PIs resistance and its regulation were also investigated. Whether CRIP1 could be a new target for MM treatment is further explored. Methods: The transcriptional level of CRIP1 was explored by RNA sequencing in plasma cells from healthy donors, newly diagnosed multiple myeloma (NDMM) and relapsed/refractory multiple myeloma (RRMM). CRIP1 overexpress and doxycycline-inducible CRIP1-shRNA cell lines were constructed to explore the role of CRIP1 in MM. The proliferation, invasion, migration, proteasome activity and autophagy were detected in CRIP1-sh/OE and control MM cells. Co-IP(Co-Immunoprecipitation) with TAP/MS (Tandem affinity purification/Mass spectrum) was performed to detect the binding of USP7, CRIP1 and PA200. Myeloma xenograft model was used to determine the role of CRIP1 to promote the proliferation of MM cells and induce BTZ resistance in vivo. Results: We found that the levels of CRIP1 were significantly increased in plasma cells of NDMM compared to the plasma cells from healthy donors (P<0.001), and further increased in patients with RRMM (P<0.05). Moreover, the high expression of CRIP1 is significantly related to the poor prognosis of MM patients(P<0.001), especially those treated with BTZ(P<0.0001). Our results in vitro and in vivo showed that CRIP1 knockdown noteworthy inhibited the cell growth, invasion, migration. MM cells were more sensitive to PIs-induced cell growth arrest when CRIP1 knockdown, and high expression of CRIP1 induces PIs resistant. Caspase-like (C-L), chymotrypsin-like (CT-L), and trypsin-like (T-L) peptidase activity of the proteasome in MM cells were significantly reduced with CRIP1 knockdown. CRIP1 Knockdown also decreased the protein level of LC3B-Ⅱin MM cell lines. Co-IP with TAP/MS analysis showed that CRIP1 formed a complex with USP7/PA200 to interaction each other. The protein level of CRIP1 decreased and the ubiquitination CRIP1 increased after P5091 treatment or with USP7 knockdown. These data suggested that the CRIP1 depletion is mediated by USP7 inhibition, and CRIP1 is the substrate of USP7.CRIP1 promotes the de-ubiquitination and stabilization of PA200 mediated by USP7 by interacting with USP7 and PA200, which in turn promotes MM cell survival and drug resistant. These results indicated that CRIP1 induces PIs resistance by forming a complex with USP7/PA200 and enhancing proteasome activity and autophagy. Inhibition of CRIP1 function by knocking down of USP7 or PSME4 in CRIP1 OE MM cell line led to a decrease of the activity of proteasome and autophagy, thus enhancing the sensitivity to PIs in intro and in vivo. Summary/Conclusion: CRIP1 plays a critical role in MM progression and PIs resistance by formation of CRIP1/USP7/PA200 complex. High level of CRIP1 is a biomarker for high-risk MM patients. CRIP1 is a potential target for MM therapy, can simultaneously inhibit proteasome activity and autophagy. Keywords: Multiple myeloma, Protein-protein interaction, Drug resistance, Proteasome inhibitor